Anodic stripping voltammetric determination of Pb, Cd, Zn and Cu in blood samples of 132 children was developed . The children's ages range was between 6 months and 6 yearsand the samples were collected from different areas of Ibb governorate (Yemen). We used anodic stripping voltammetry for analyzing and determination of cited metals in the blood of all children. Large variation in the results have been correlated to the area inhabited, age differences and other factors.
It was found that areas, which have environmental struts such as waste solid and waste water, had more quantities of these metals in blood samples in comparison with other safer sites. This is may be due to the lower concentration of Cd and Pb in samples.
Also the results indicate that both Pb, Cd found in high mean concentration in blood samples of males children than in females children similarly the effect of age indicate that all younger children (between 6 months and 1 year) had shown comparatively lesser quantity of these metals in comparison with elder children (between 4 - 6 yeas).
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Keywords: Anodic stripping voltammetry, determination, heavy metals, children, blood.
Environmental pollution nowadays is a big concern. Anthropogenic activities have altered the environment significantly throughout the world like mining, industry and agriculture (Wang et al, 2008). Environmental contaminants are widely distributed in air, water and soils and, therefore, they will have an effect on the trophic chain (vegetables, animals and men) (Tiller, 1989). Heavy metals are of considerable behavior (Omgbu & Kokogbo, 1993).
The population of women exposed to lead either at work or in their environment show a correlation between high lead content in the blood and the low birth weight of their children (Uzych, 1985). It was also shown that lead toxicity may cause mental and psychomotor retardation of children without encephalopathy (Macdonell & Campdell-Handstone, 2000; Sallmen et al, 1992). The definition of lead poisoning (i.e. â‰¥ 10 Âµg/dl) adopted by many international institutes including the CDC (Centers for Disease Control and Prevention, USA) was lowered to a blood lead level of 10 Î¼g/dl in 1991 due to the rapid decline in environmental and occupational lead exposure in most developed countries (Prikle et al, 1994), Therefore, blood lead level <10 Âµg/dl is now universally accepted as the low-lead exposure threshold (Markowitz et al, 1997)
Cadmium toxicity may cause premature birth, low birth weight and even disturb production of chronic gonadotrophin by the placenta and impair development of the near born vascular system (Baird, 1993) . It was demonstrated that cadmium effects on the genes were involved with growth regulation of initiated cells (Fang et al, 2002) .
In 1980, World Health Organization (WHO) stated that a value of 10 Âµgl-1 of Cadmium in whole bloods should be accepted as tentative non-adverse effect level and the level of lead should be restricted to 3mg/week (0.05mgKg-1 of body weight) for adult; no corresponding values for children were suggested, which clearly indicate that children should be kept as free from these metals as possible, because children have a great potential for lead and Cadmium exposures and are uniquely susceptible to their toxic effects (Krishnamurty,1991).
Since the blood brain barrier is not fully developed in young children, their developing nervous system accumulates the ingested lead resulting in neurobehavioral barrier. As the lead crosses the placental barrier, the developing fetus is also at high risk of lead poisoning from mother's blood (Popko et al, 2003). Moreover the normal level of cadmium in blood is under 5Âµgl-1 but acute toxic effects observed when this level exceed 50Âµgl-1, (Pais & Jones ,1997). Greater prevalence of iron deficiency in young children also increases the gastrointestinal absorption of lead (Albbasi& Rajindra, 1998). However in case of cadmium placenta acts as a fairly effective barrier and hence the new born are virtually free of cadmium. It was also shown that accumulation in the body increases with age and at the age of 50, non-occupationally exposed people may have 10-50 mg body burden of cadmium (Krausma et al.2004a).
Zinc is an important nutritive factor as well as a co-factor for many metalloenzymes. It is naturally present in blood at a high level 10-5 M (Krausma, 2004b), and necessary for the growth and division of cells, especially during the states of life when growth rates are high (Hurley & Sandswenrton, 1966, Krachler et al, 1999).
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Copper indicates the mobilization in the mother's body during pregnancy. Its deficiency may cause disorders (Uauy et al, 1998). Copper in blood is bound to serum albumin (Kandzierska, 1989). The main physiological processes in which copper participates in the formation of blood and the utilization of iron in hemoglobin synthesis, the synthesis and cross-linking of elastin and collagen in the aorta and major blood vessels, etc. So both copper and zinc are known to be beneficial for humans when present in low concentration only; but Lead and Cadmium are known to be toxic even at very low concentration (Zhao et al, 1990; Osofasky & Jowelt, 2001).
The level of lead, cadmium, zinc and copper in the blood is considered as an index of biologically active metal in the body, reflecting also the environmental expose of a population. The concentration of these metals in the blood is a significant factor for child health (Mahajan et al, 2005) .
Electrochemical stripping analysis is a very important and widely used tool for the trace determination of various elements and compounds. Major advantages include low detection limits coupled with good selectivity and reproducibility due to the in situ pre-concentration step. This is especially true for anodic stripping voltammetry (ASV) has attracted considerable attention for the determination of trace and ultra-trace metals because of their unique feature high sensitivity, accuracy, operation and low costs of instrumentation (Wang, 1985).
Differential Pulse Stripping Voltammetry (DPSV) is relatively inexpensive and is one of the most sensitive and selective techniques in the determination of trace amounts of metals at natural levels ( Mahajan et al 2002 , Celik & Oehlenschlager , 2007, Behrooz et al, 2009).
In the present work we study the use of differential pulse anodic stripping voltammetry for the evaluation and determination of trace metals in child blood whose age between 6 months and 6 years, in some areas of Ibb governorate, Yemen.
Materials and Methods:
Stripping voltammetric experiments were carried out with a Metrohm 746VA (Herisau, Switzerland). Trace Analyzer connected to a Metrohm 747VA multimode electrode used in the hanging mercury drop electrode (HMDE) regime. A platinum rod and saturated Ag/AgCl electrodes were used as auxiliary and reference electrodes respectively. pH was measured with a digital pH-meter JENWAY, model 3310. Dissolved oxygen was removed from the samples by purging with purified nitrogen (99.99%) through the measuring vessel for 5 min. During the experiments, nitrogen was passed over the solution to prevent oxygen interference.
The tartaric acid, CH3COOH, CH3COONa, Pb(NO3)2, Cd(NO3)2, Cu(NO3)2, Zn(NO3)2 and HNO3 used were from Merck, Darmstadt ,Germany. De-ionized water was used to prepare all solutions. The metal stock solutions ( 1g/L) were prepared in 0. 005 M HNO3. The working standard solutions were prepared daily by suitable dilution of this stock solution in the required matrix. The acetate buffer solution was 0.2 M and pH 4.7 containing 0.2 M tartaric acid , which served as a supporting electrolyte. All glassware were stored in 8M nitric acid for 1 week and rinsed thoroughly with de-ionized water.
Sampling and sample pretreatment:
From October 2007 to may 2008, one hundred and thirty two (132) Yemeni children, aged between 6 months and 6 years, were collected at the governmental hospitals in Ibb city for estimating the impacts of water, food and atmospheric pollutions on the human health. Children being more sensitive to the pollutants and more at risk as compared to adults. Their bloods are considered as an index of biologically active metal in the body reflecting direct effect of pollutions on populations. Blood samples were analyzed for four heavy metals of Cd, Pb, Zn and Cu. Data of hemoglobin, malnutrition and weight measurements of the same children was collected in order to correlate these parameters with the occurrence of heavy metals in blood samples.
2.0 ml blood samples were taken from each child with special care by vein puncture sing disposable syringes and needles and placed into heparinized pretreated clean polypropylene tubes. The sample were then digested with 4ml of 70% nitric acid and 1ml of 70% perchloric acid 60% then add few drops hydrogen peroxide (30 %), heating to dryness. The residue was wet with 1 mL nitric acid 65 % and 9 ml distilled water, and then the crucible was boiled on a hot plate to the complete dissolution of the residue . The sample was then quantitatively transferred to 25ml volumetric flask and diluted to the mark.
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A 0.5ml of digested sample was directly transferred to the voltammetric cell containing 20ml of ammonium acetate buffer (pH 4.7). The stripping voltagram for zinc, cadmium, lead and cppper was recorded after arrest period of 10 s after stirrer stopping. The potential was swept using deferential - pulse anodic stripping voltammetry (DPASV) selecting preconcentration time of 240 s for Cd , Pb and Cu and 120 s for Zn, a scan rate of 60 mVs-1, and a pulse amplitude of 50 mV. The standard additions technique was used to give the concentrations of Cd , Pb ,Cu and Zn simultaneously when a sweep potential was applied between -1200 mV and 200 mV ( vs. Ag/AgCl electrode).
The stripping voltammetric measurement was repeated (each measurement must be done at least twice), 100 ÂµL of standard metal solutions was added (standard addition calibration method), and the new voltagram was recorded again. Voltagram of these elements is shown in Figure 1. The concentrations were calculated after the second standard addition, using linear regression method.
Statistical analysis of data was carried out using SPSS (version 16) statistical package program. The means and standard deviations and prevalence were obtained by descriptive statistics. Data were analysed using the one way analysis of variance (ANOVA) followed by T- test to assess difference of continuous variable between two or more groups. Statistical signiï¬cance was assigned for p-values less than 0.05.
Results and Discussion:
The calibration carves were linear in the concentrations range of 50-6000 Âµgl-1 with a correlation coefficient lies between 0.9955-0.9992 for the four elements. Based on the calibration curve, the limits of detection were also determined. The limits of detection are the analyte concentration giving a signal equal to the blank signal, three standard deviations (Farrant, 1997). The limits of detection were 2.53Âµgl-1Pb, 0.18Âµgl-1 Cd, 260.40Âµgl-1Cu and 311.46 Âµgl-1Zn. The precision of the proposed method was estimated by calculating the relative standard deviation (RSD) which were 3.51% , 4.18%, 2.79% and 3.24%, respectively.
Effect of location:
It has already been well documented by many workers that living locality has a great impact on occurrence of heavy metals in blood samples, locations with industries waste solid and other such sources that emit various metals to the inhabitants through a variety of routes (Mahajan et al, 2004). In our study, we had chosen four sites from different locations of Ibb governorate Fig.2. Table.1 shows the concentration of heavy metals in blood samples collected from different locations of Ibb governorate. It can be seen from Table.2 that all the samples mean that were taken from Almashana and Refibb 50.56 Â± 16.52, 61.66 Â± 26.12 Âµgl-1 for Pb and 3.90 Â± 1.12, 5.14 Â± 0.73 Âµgl-1 for Cd, The results showed significantly higher (p < 0.05) of lead and cadmium then the other areas. This was due to the presence of metal based pollution of waste solid, wastewater (Matloob et al, 2001, Alhemiary, 2009, Al-sabahi et al, 2009), main high way that discharge large amount of lead and cadmium. Containing effluent. However, quantities of zinc and copper in these tow locations showed an inverse trend. This could be related to tow level of hemoglobin malnutrition in these children, Table.2. It s seen that the children with malnutrition or with hemoglobin level less than 6mg/l have low level zinc and copper. Some workers (Kandzierska, 1989) found similar types of correlations; However, it was difficult to correlate the effects of pollutants and concentrations of hemoglobin and malnutrition. It can be seen from the Table.1 that samples from Aldehar and Alodian, which are comparatively safer, showed lesser concentration of cadmium and lead comparison to Almashan and Refibb. However, higher concentration of zinc and copper at Aldehar and Alodian (2391.85 Â± 780.50 , 2258.54 Â± 976.70 Âµgl-1 for Zn and 2740.27 Â±1201.37, 2320.12 Â± 946.32Âµgl-1 for Cu showed significantly higher (p < 0.05), can be related to the fact that all these samples were having the largest concentration of hemoglobin and they were having the largest body weights ( Turgut et al,2007). So it can be said that all these samples which were taken from anemic and malnourished children had shown lower concentration of zinc and copper in comparison to those that were normal. It is well documented that a small increase in the lead levels in the air can lead to a higher concentration in blood level of lead (Chamberlin, 1983). Based on epidemiological and experimental data comparative suggested that an increase in air lead of 1Âµgl-1 would result in increase in blood lead of 15-30 Âµgl-1 .Similar results are observed for Aldehar showing higher concentration of lead probably due to higher vehicular population and these higher amount of lead in the air.
Effect of sex:
Effects of sex on the uptakes of these metals are presented in Table.3. Though, the variation appears small with regard to lead and cadmium among both sexes, a larger variation can be seen with zinc and copper. Both lead, cadmium copper and zinc are found in higher concentration in males in comparison to female children 51.36Â± 21.26, 4.59 Â± 1.37,2078.23 Â± 765.8 and 2514.6Â± 1107.33Âµgl-1 respectively. The results of our study metals showed significantly (p < 0.05) that males tend to have higher blood than females. All males children showed higher concentration of copper than the female children, and this may be due to lower levels of hemoglobin and lesser number of red blood cells in female children. Many workers (Alsaleh &Taylor, 1994; Mahajan &Walia, 2005) obtained similar results.
Effect of Age:
All the 132 samples were categorized into four different age groups as shown in Table. 4. It is evident from Table. 4 that all the children whose age were between 6 months -1 year and 1-2 years have lower mean concentration of all these metals except lead (35.83 Â± 13.37, 22.28 Â± 4.46 Âµgl-1). The results showed significantly higher (p < 0.05) as compared to the children of other ages. The children aged between 4-6 years show maximum mean concentration of metals except lead. The results show that there is a direct correlation between the age and concentration of the metals in the blood and this may be due to the longer length of expose in older children (Pitman & Hilts,2007, CDC, 2007). Exception of lead can be related to other factors, most of the samples from aged between 4-6 years were taken from Aldehar and Almshana where a heavy vehicular pollution could be responsible. It was also known that lead passes through the placenta easily and fetal blood has almost the same blood lead concentration as maternal blood (Chamberlin, 1983). Therefore, exceptionally higher concentration of lead in the blood of all the infants from Almshana, Refibb and Aldehar may be because of higher exposure of their mother's to the vehicular pollution. However, The mean of cadmium in the infants ages between 6 months -1 year and 1-2 years remained lower 3.43 Â± 1.72, 2.43 Â± 1.05 Âµgl-1 because in this case placenta acts as a fairly effect barrier to cadmium and hence new born are virtually free from cadmium. Accumulation of cadmium in the body increase with age, as shown by other workers (Alsaleh &Taylor,1994; Hans & Sigel,1998). It is also known that cadmium is always found in association with zinc, so all samples show higher concentration of cadmium but lower concentration of zinc (/Ø§Ø¨ØØ§Ø«Ø¹Ù†Ø§ØµØ±/Journal of Allergy and Clinical Immunology - Record.htm1.htm"Wilhelm et al, 1994, Teliá¹§man et al,2000).
Although, there are a direct correlation of concentration of blood levels of metals with age because of higher exposure. It can be concluded that all the younger children, because of their lesser exposure are at lower risk to the pollutants than their elder counterparts and all those, which are living areas pollutant, are increasing their bloods metal content with passage of time.
We conclude that, all those children who are residing in and around pollution areas (waste solid, wastewater and dense vehicular) at higher risk of the environmental stress than those from safer sites. The increase can accumulating nurture of heavy metals. The may reach a dangerous level if an immediate measures are not taken to shift the location.